Capillary Electrophoresis (CE), Micro-Column Chromatography and Capillary Chromatography, more particularly High Performance Liquid Chromatography (HPLC), are well known techniques for liquid-phase chemical separation in small volume chemical analysis. There exists a great demand for improvements of the instrumental set-ups for these separation techniques, in order to achieve better separation results or a so-called higher number of theoretical plates, faster analysis times and lower reagent consumptions. Among the various aspects contributing to the overall performance in Capillary Electrophoresis (CE) and Micro-column and Capillary Chromatography (HPLC), the detection, which is most commonly performed by optical methods, is a very important one. There exists a great demand to be able to detect the substances of interest within nano-liter or even pico-liters volumes. Detection represents thus the main obstacle in the quest for higher miniaturization for small volume chemical analysis.
In Capillary Electrophoresis for example, to preserve the spatial profile of the eluting substances and, considering that the total volume of the separation stage, including the detection arrangement, is usually less than a milliliter, dead-volumes must be avoided. Under these circumstances it was found that meaningful results can only be achieved by on-column detection. This is also the case in HPLC. From the prior art several arrangements for on-column optical detection are known. These include detection arrangements for absorption, fluorescence, and refractive index measurements, as are described for example in N. J. Dovichi, Rev. Sci. Instrum. 61, 3653 (1990). There is, however, a growing demand to improve the sensitivity of those detection systems, to reduce their detection volumes while at the same time retaining the instrumental sensitivity.
Laser Induced Fluorescence (LIF) detection is, to date, considered to be one of the most sensitive detection methods for chemical separations in capillary tubes. However, even fluorescence detection methods based on conventional arc or filament lamps as excitation sources are suitable, although they are less sensitive.
The main advantages of lasers, as compared to conventional excitation sources, reside in their high intensifies and good spatial properties. However, both, lasers and conventional lamps display high fluctuations of their light intensities, which is very undesireable for detection purposes. Intensity fluctuations of the excitation source have a negative impact on fluorescence detection arrangements because they manifest in both, the fluorescence signal (S) and in the background noise (N). This latter because of the unavoidable amount of scattering light, which reaches the usually employed photomultiplier.
Fluorescence detectors operating without scattered light are known as background-free detectors. The baseline noise of these detectors is dominated by shot-noise. Background-free detectors are not uncommon for gas phase detection but they are more difficult to realize in the liquid phase, and it is even more difficult in the presence of liquid filled narrow bore capillary tubes. The difficulties arise primarily from the fact, that scattering light is produced at the four unavoidable optical interfaces in the light propagation media at the measuring zone. These difficulties are described in more detail for example in A. E. Bruno, B. Krattiger, F. Maystre and H. M. Widmer, Anal. Chem., 63, 2689 (1991). The four optical interfaces arise at the walls of the capillary tube and are the interfaces air/FS, FS/buffer, buffer/FS and FS/air, wherein FS refers to the capillary tube material, which normally is fused silica or another type of glass.
It is to be noted that scattering light is not only a major problem for fluorescence detectors, but also limits the resolution of other detector arrangements, such as for example for absorption, and refractive index measurements. In these detector arrangements, as well as in the ones for fluorescence measurements the baseline noise constitutes a major limiting factor for the application of the respective methods. The ultimate sensitivity of optical detectors employed is often limited by noise and drifts caused by the thermal expansions of the materials involved, by vibrations and Schlieren effects in the light propagation media, which starts at the light source and ends at the surface of the photoelectric detector. These noise and drift sources are mainly generated at the various optical interfaces, where reflection and refraction takes place, and, they are more pronounced when the interfaces encountered are not flat but have a curvature, like in the case of lenses or round capillaries.
In the past solutions have been proposed to minimize the amount of scattering light reaching the photosensitive devices such as the photosensitive detector and the photomultipliers. One of these approaches is described for example in N. J. Dovichi, Rev. Sci. Instrum. 61, 3653, (1990). The approach consists in the elimination of all optical interfaces in the area of the measuring zone. The socalled "windowless cell" is known as "sheath flow cuvette" and is arranged at the end of the capillary tube. Unfortunately this approach is not particularly easy to implement and only few optical detectors can be constructed in the proposed manner.
In indirect fluorescence detection arrangements the baseline noise is almost entirely due to the noise of the fight source. In indirect fluorescence the solvent or buffer used in the separation is doped with a low concentration of a fluorophore. Elution of non-fluorescent ionic analytes, instead of diluting the solvent, displaces the fluorescent dye. The detection of the substances of interest is accomplished by monitoring the decrease in the fluorescence (i.e. a fluorescence dip) due to a decrease in the concentration of the buffer or solvent. From the outlined detection principle it is easily conceivable that intensity fluctuations of the excitation light source are detrimental to the measurements.